Chronic respiratory infections with Pseudomonas aeruginosa are the primary causes of high morbidity and mortality in cystic fibrosis (CF). We have recently developed a unique pulmonary infection mouse model that depends on the artificially generated P. aeruginosa aerosol to cause a uniform whole-lung infection in mice. The focus of this revised proposal is to test a group of 90 clinical CF isolates of P. aeruginosa for innate lung clearance, cytokine profiles and histopathology in this aerosol infection model. The hypothesis to be tested here is that the hypervariable chromosomal restriction fragment length polymorphisms (RFLPs) of the clinical CF sputum isolates may contribute to i) variations in bacterial respiratory colonization capacity, ii) altered levels of cytokine production by the host, and iii) the different outcomes of lung pathology. This is based on our following recent observations. First, we have applied the technique of pulsed field gel electrophoresis (PFGE) to analyze a collection of 90 clinical CF isolates for their genomic profiles. We have established a database composed of 75 unique Spe-I restriction digest PFGE profiles. Out of 90 strains tested, we identified one isolate CF32 that had identical Spe-I, Xba-I and Dpn-I digest PFGE patterns as P. Aeruginosa PAO1, a standard reference strain of a wound origin. Secondly, we passed PAO1 and 3 other CF isolates including the PAO-1 like CF isolate through the aerosol infection system to test for the pulmonary clearance and production of tumor necrosis factor (TNF)-a. PA01 and CF32 showed a similar pattern of lung clearance and TNF-a induction in the C57BL/6J and BALB/cJ mice. However, the other 2 CF isolates were more resistant to the clearance by the BALB/cJ mice. One isolate (CF45) caused a significant induction of TNF-a by the murine lungs. These results indicate that the genomes of the CF isolates are highly diversified, and the genomic diversity may affect their intrinsic biological properties. More importantly, it's feasible to use the aerosol apparatus to assess the remaining CF isolates for their virulence properties. The future directions that this project may lead to include i) investigations of the novel Pseudomonas genes induced due to lung colonizations; ii) exploration of the novel DNA fragments missing in the PAO1 genome but present in a subset of the CF isolates; iii) DNA immunization and testing for protection, and iv) evaluation of some selected CF isolates in the aerosol mouse model. By achieving the research objective of this Academic Research Enhancement Award (AREA) that is to establish and infection database for the CF isolates in the aerosol model, we will have an essential base of knowledge from which to prepare a future R01 application to investigate the novel virulence mechanisms associated with the clinical CF isolates of P. aeruginosa.